Novel Requirements for Akt1 in T Cell Commitment

摘要 AKT1、Akt2和AKT3是丝氨酸/苏氨酸激酶家族的成员，该家族是PI3K的关键效应因子。这个 AKT家族具有促进细胞存活、糖代谢和增殖的作用。 多种细胞类型，包括发育中的T细胞。胸腺细胞不同程度地表达所有Akt亚型。 单个或多个Akt基因的去除会导致T细胞发育的差异阻断。三个都失去了 AKT亚型导致早期发育缺陷，原因是CD4-CD8-Double存活率降低 阴性(DN)胸腺细胞。Akt2或AKT3单独表达足以维持糖尿病肾病的存活率；然而， DN3分化和DN4胸腺细胞增殖在后期仍有缺陷，细胞存活 发育阶段。这些不同的表型表明，不同的Akt亚型调节不同的 功能或不同剂量的Akt活性是促进特定阶段细胞存活所必需的， 增殖分化。由于目前还没有关于异构体特定功能的证据，我们将我们的 注意调节Akt活性剂量的特征机制。表面受体，包括 (前)T细胞受体和细胞因子受体激活PI3K产生PIP3。PIP3和PIP3之间的相互作用 Akt的Pleckstrin同源(PH)结构域需要将Akt招募到质膜上 激活。因此，对PI3K活性和PIP3活性的精细控制可以直接调节Akt的激活水平。 该应用程序解决了间接控制Akt的一种日益重要的替代机制 活性：通过产生IP4，一种结构类似于PIP3的可溶性肌醇多聚磷酸。IP4是 在(前)TCR刺激后产生，并与PIP3竞争结合Akt PH结构域。 缺乏IP4的胸腺细胞在DN3和DN4表达preTCR后过度激活Akt 各阶段。引人注目的是，Akt多动导致β选择加速，过早地重新编程为糖酵解 Notch依赖的新陈代谢和丧失。这导致我们提出以下全球假设：1)前TCR- 诱导的IP4生成限制Akt依赖的代谢重编程和增殖以确保 足够的Notch信号和2)前TCR和Notch协同信号是T细胞谱系所必需的 规格。这项研究的成功完成将改变我们目前对T细胞谱系的理解 规范通过揭示1)在实施凹槽检查点和在 共刺激Notch功能，2)控制胸腺细胞的一种新的前TCR反馈机制 通过调节Akt活性和代谢重新编程的增殖/成熟；以及3)非规范的 激活依赖于TCF1的转录以确定T细胞承诺的途径。

ABSTRACT Akt1, Akt2 and Akt3 are members of a family of serine/threonine kinases that are key effectors of PI3K. The Akt family has been well characterized to promote cell survival, glucose metabolism and proliferation in many cell types including developing T cells. Thymocytes express all Akt isoforms to varying degrees. Ablation of single or multiple Akt genes results in differential blocks in T cell development. Loss of all three Akt isoforms leads to an early developmental defect due to decreased survival of CD4–CD8– double negative (DN) thymocytes. Expression of Akt2 or Akt3 alone is sufficient to preserve DN survival; however, DN3 differentiation and DN4 thymocyte proliferation remain defective along with survival of cells at later developmental stages. These distinct phenotypes suggest either that different Akt isoforms regulate distinct functions or that different doses of Akt activity are required to promote stage-specific cell survival, proliferation and differentiation. With no current evidence for isoform-specific functions, we focused our attention on characterizing mechanisms for regulating the dose of Akt activity. Surface receptors, including (pre-)T cell receptor and cytokine receptors, activate PI3K to generate PIP3. Interactions between PIP3 and the Pleckstrin homology (PH) domain of Akt are required to recruit Akt to the plasma membrane for activation. Thus, fine control of PI3K activity and PIP3 availability can directly regulate Akt activation levels. This application addresses an increasingly important alternative mechanism for indirectly controlling Akt activity: via generation of IP4, a soluble inositol polyphosphate that is structurally similar to PIP3. IP4 is generated following (pre)TCR stimulation and competes with PIP3 for binding to the Akt PH domain. Thymocytes deficient in IP4 activate Akt excessively following expression of preTCR at the DN3 and DN4 stages. Strikingly, Akt hyperactivity leads to accelerated β selection, premature reprogramming to glycolytic metabolism and loss of Notch dependency. This leads us to propose the global hypotheses that 1) preTCR- induced IP4 generation restricts Akt-dependent metabolic reprogramming and proliferation to ensure adequate Notch signaling and 2) preTCR and Notch cooperative signaling is required for T cell lineage specification. Successful completion of this study will shift our current understanding of T lineage specification by revealing 1) an unexpected requirement for preTCR in imposing a Notch checkpoint and in co-stimulating Notch function, 2) a novel preTCR feedback mechanism that controls DN thymocyte proliferation/maturation via tuning of Akt activity and metabolic reprogramming; and 3) a non-canonical pathway for activating TCF1-dependent transcription to specify T cell commitment.